Transgenic animals

Abstract

The present invention provides improved methods and compositions for the generation of transgenic non-human animals. The present invention permits the introduction of exogenous nucleic acid sequences into the genome of unfertilized eggs (e.g., pre-maturation oocytes and pre-fertilization oocytes) by microinjection of infectious retrovirus into the perivitelline space of the egg. The methods of the present invention provide an increased efficiency of production of transgenic animals with a reduced rate of generating animals which are mosaic for the presence of the transgene.

1. A method for introducing a polynucleotide contained within the genome of a recombinant retrovirus into the genome of a non-human mammalian zygote, comprising:
 a) providing a plurality of non-human mammalian zygotes, each having a plasma membrane and a zona pellucida, said plasma membrane and said zona pellucida defining a perivitelline space; 
 b) providing an aqueous solution, which has been rendered cell-free by ultracentrifugation, wherein said cell-free aqueous solution comprises a polynucleotide contained within the genome of a recombinant retrovirus; 
 c) introducing said solution comprising said polynucleotide contained within the genome into said perivitelline space by injection through a needle, under conditions which permit the integration of said polynucleotide contained within the genome of a recombinant retrovirus into the genome of said zygotes, such that said polynucleotide is stably maintained, thereby integrating the polynucleotide into the genome of at least twenty percent of said zygotes; and 
 d) culturing said zygotes which have integrated the polynucleotide in their genomes. 
 
     
     
       2. The method of  claim 1 , wherein said polynucleotide contained within the genome of a recombinant retrovirus encodes a protein of interest. 
     
     
       3. The method of  claim 2 , further comprising the step of selecting a cultured zygote from said zygotes which have integrated the polynucleotide in their genomes and transferring said zygote into a non-human mammalian recipient that is hormonally synchronized to simulate early pregnancy, thereby giving a transferred embryo. 
     
     
       4. The method of  claim 3 , further comprising the step of allowing said transferred embryo to develop to term. 
     
     
       5. The method of  claim 4 , further comprising the step of identifying at least one transgenic offspring. 
     
     
       6. The method of  claim 5 , wherein said transgenic offspring expresses said protein of interest. 
     
     
       7. The method of  claim 1 , wherein said recombinant retrovirus comprises a Moloney murine leukemia virus long terminal repeat. 
     
     
       8. The method of  claim 1 , wherein said recombinant retrovirus comprises a heterologous membrane-associated protein. 
     
     
       9. The method of  claim 8 , wherein said heterologous membrane-associated protein is a G glycoprotein selected from a virus within the family Rhabdoviridae. 
     
     
       10. The method of  claim 9 , wherein said G glycoprotein is selected from the group consisting of the G glycoprotein of vesicular stomatitis virus, Piry virus, Chandipura virus, Spring viremia of carp virus, Rabies virus, and Mokola virus. 
     
     
       11. A method for introducing a polynucleotide contained within the genome of a recombinant pseudotyped retrovirus into the genome of a non-human mammalian zygote, comprising:
 a) providing a non-human mammalian zygote having a plasma membrane and a zona pellucida, said plasma membrane and said zona pellucida defining a perivitelline space; 
 b) providing an aqueous solution, which has been rendered cell-free by ultracentrifugation, wherein said cell-free aqueous solution comprises a polynucleotide contained within the genome of a recombinant pseudotyped retrovirus; and 
 c) introducing said solution comprising said polynucleotide contained within the recombinant retrovirus into said perivitelline space by injection through a needle, under conditions which permit the integration of said polynucleotide contained within the pseudotyped retrovirus into the genome of said zygote, such that said polynucleotide is stably maintained. 
 
     
     
       12. The method of  claim 11 , wherein the introduction of said polynucleotide contained within the genome of a pseudotyped retrovirus results in integration of the polynucleotide in at least twenty percent of the zygotes into which the polynucleotide is introduced. 
     
     
       13. The method of  claim 11 , wherein said polynucleotide contained within the genome of a pseudotyped retrovirus encodes a protein of interest. 
     
     
       14. The method of  claim 13 , further comprising the step of transferring said zygote into a non-human mammalian recipient that is hormonally synchronized to simulate early pregnancy, thereby giving a transferred embryo. 
     
     
       15. The method of  claim 14 , further comprising the step of allowing said transferred embryo to develop to term. 
     
     
       16. The method of  claim 15 , further comprising the step of identifying at least one transgenic offspring. 
     
     
       17. The method of  claim 11 , wherein said pseudotyped retrovirus comprises a Moloney murine leukemia virus long terminal repeat. 
     
     
       18. The method of  claim 16 , wherein said transgenic offspring expresses said protein of interest. 
     
     
       19. The method of  claim 11 , wherein said pseudotyped retrovirus comprises a heterologous membrane-associated protein. 
     
     
       20. The method of  claim 19 , wherein said heterologous membrane-associated protein is a G glycoprotein selected from a virus within the family Rhabdoviridae. 
     
     
       21. The method of  claim 20 , wherein said G glycoprotein is selected from the group consisting of the G glycoprotein of vesicular stomatitis virus, Piry virus, Chandipura virus, Spring viremia of carp virus, Rabies virus, and Mokola virus.